A variety of nucleic acid-based therapeutics designed to regulate aberrant gene expression associated with human disease are currently under development. Such strategies include, but are not limited to “antisense” therapy and RNA interference (“RNAi”). Antisense therapy comprises administration or in situ generation of nucleic acid molecules (i.e., RNA or DNA, or modified derivatives thereof) which specifically bind to cellular RNA (i.e., mRNA) or genomic DNA, thereby inhibiting expression of a specific protein by inhibiting its transcription and/or translation. The binding may be by conventional Watson-Crick base pairing, by specific interactions in the major groove of the double helix, or by still other types of molecular interaction (i.e., Hoogsteen base pairing). Antisense RNAs which are complementary to the 5′ untranslated region of an mRNA up to and including the initiation codon work most efficiently at inhibiting translation. However, sequences complementary to the 3′ untranslated sequences of mRNAs inhibit translation of mRNAs as well. Therefore, antisense RNAs complementary to either the 5′ or 3′ untranslated regions of a gene can be used to inhibit translation of endogenous mRNA.
RNA interference (RNAi) is an evolutionarily conserved process for specific silencing of gene expression. The discovery that synthetic short interfering RNAs (siRNAs) of ˜19-29 bp can effectively inhibit gene expression in mammalian cells and animals without activating an immune response has led to a flurry of activity to develop these inhibitors as therapeutics. Inhibition is caused by the specific degradation of the messenger RNA (mRNA) transcribed from the target gene. In greater detail, RNA interference describes a process of sequence-specific post-transcriptional gene silencing in animals mediated by so called “siRNAs” (Fire et al., “Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans,” Nature 391:806-11 (1998)). Chemical stabilization of siRNAs results in increased serum half life, suggesting that intravenous administration may achieve positive therapeutic outcomes if delivery issues can be overcome. Consequently, synthetic short interfering RNAs (siRNAs) have emerged as an important tool for post-transcriptional gene silencing in mammalian cells and live animals.
The natural function of RNAi appears to be protection of the genome against invasion by mobile genetic elements such as retrotransposons and viruses which produce aberrant RNA or dsRNA in the host cell when they become active. The process of post-transcriptional gene silencing is therefore believed to be an evolutionarily-conserved cellular defense mechanism present in the majority of mammalian cell types and is used to prevent the expression of foreign genes such as those derived from infection of viruses. This assumption is further strengthened by the observation that RNAi in animals, and the related phenomenon of post-transcriptional gene silencing (PTGS) in plants, result from the same highly conserved mechanism, indicating an ancient origin.
RNA interference involves the processing of a double-stranded RNA (dsRNA) into shorter units (called siRNAs) that guide recognition and targeted cleavage of homologous target messenger RNA (mRNA). The first step of the process involves a dsRNA endonuclease activity (ribonuclease III-like; RNase III-like) that processes dsRNA into smaller sense and antisense RNAs in the range of 19 to 25 nucleotides long, producing the short interfering RNAs (siRNAs). That RNase III-type protein is termed “Dicer”. In a second step, the siRNAs produced combine with, and serve as guides for, a different ribonuclease complex called the RNA-induced silencing complex (RISC), which recognizes and cleaves the target homologous single-stranded mRNAs.
While this technology has revolutionized research, however, inability to deliver siRNAs and other nucleic acid-based therapeutics systemically to cells remains the largest obstacle for in vivo clinical applications of such therapeutics. Although delivery of siRNA across plasma membranes can be achieved with vector-based delivery systems, high pressure intravenous injections of siRNA or chemically modified siRNAs such as cholesterol conjugated siRNAs, those technologies have intrinsic limitations.
RNA interference (RNAi) has enormous therapeutic potential. Specific gene silencing using small interfering RNA (siRNA) can disrupt virus reproduction and turn off genes related to metastatic cancer or aberrant metabolic processes, such as Alzheimer's disease. The commercial potential for this technology has yet to be realized and, in certain respects, is linked to development of viable methods for systemic delivery and targeting of siRNA.